Protein identification refers to determining the type of protein in the sample being examined, and is most commonly performed by mass spectrometry. There are many types of protein samples that can be detected in this case.
The common samples currently available are:
Cell Lysates:
Cell lysates serve as a fundamental sample type for LC-MS/MS analysis. Through cell lysis, proteins are released for subsequent chromatographic separation and mass spectrometry. The method provides a holistic view of the cellular proteome, enabling a comprehensive analysis of the proteins present in the sample. However, potential challenges include contamination from cellular debris, emphasizing the need for careful selection of lysis buffers to optimize protein extraction efficiency.
Tissue Homogenates:
Tissue homogenates involve the disruption of cellular structures to release proteins for analysis. This sample type is valuable for studying organ-specific proteomes, offering insights into the in vivo protein composition of tissues. Researchers must, however, be mindful of the homogenization method, as it can impact protein integrity. Additionally, the presence of endogenous enzymes poses a risk of protein degradation.
Plasma/Serum Samples:
Plasma or serum samples are commonly used in LC-MS/MS for biomarker discovery and disease studies. These samples reflect systemic changes in protein expression and allow for minimally invasive sampling. Challenges include the abundance of high-abundance proteins (e.g., albumin), which may mask the detection of low-abundance proteins. Rigorous sample preparation is crucial to remove interfering substances and enhance result accuracy.
Urine Samples:
Urine, a non-invasive sample type, is suitable for biomarker discovery and renal-related studies. It is easy to collect and contains proteins reflective of renal function and systemic changes. However, challenges such as dilution effects and variability in protein concentration must be addressed. Careful sample handling is essential to ensure the reliability of results.
Protein Complexes/Purified Proteins:
Analysis of purified protein complexes or individual proteins is another facet of LC-MS/MS. This approach allows for focused analysis of specific proteins or complexes, making it ideal for functional studies. However, researchers should be aware that the obtained results may not fully represent the complexity of biological samples. Sample purity is a critical consideration in this context.
Here are some special samples:
IP-derived Samples:
Immunoprecipitation involves using antibodies to isolate specific proteins from samples containing a mix of proteins. If the targeted protein is unknown or not previously predicted, mass spectrometry can be employed for identification. This method, while effective, separates only one protein per IP, limiting its capacity for comprehensive analysis.
CoIP-derived Samples:
Co-immunoprecipitation captures protein complexes formed within a sample by utilizing antibodies against a target protein. Since these complexes represent real interactions within the sample, CoIP provides a more accurate reflection of protein-protein interactions. Verification through Western blotting (WB) can be conducted if prior predictions of interacting proteins exist. Otherwise, mass spectrometry can be applied for identification of unknown interacting proteins.
DNA Pull Down-derived Samples:
DNA pull-down studies known DNA sequences to identify unknown proteins that bind to them. This method is particularly useful for researching gene regulatory elements and transcription factor relationships. Biotin-labeled known DNA sequences are incubated with the protein solution, forming a biotin-DNA-protein complex. Magnetic beads with streptavidin are then employed to separate this complex, followed by LC-MS/MS for the identification of proteins bound to the target DNA.
For instance, studying transcription factors binding to the promoter of Gene A can be accomplished through DNA pull-down. After amplifying and verifying the promoter region of Gene A, labeled DNA is incubated with nuclear proteins, enriched using magnetic beads, and subjected to LC-MS/MS to identify the interacting proteins.
RNA Pull Down-derived Samples:
RNA pull-down, similar to DNA pull-down, studies known RNA sequences and their associated proteins. Biotin-labeled RNA sequences are incubated with the protein solution, forming a biotin-RNA-protein complex. This complex is then separated using streptavidin-coated magnetic beads, followed by LC-MS/MS for identifying proteins bound to the target RNA.
In protein identification submission guidelines, certain sample types such as immunoprecipitation (IP) and pull-down samples are less commonly encountered, while gel strips, magnetic bead samples, and protein solutions are more prevalent. This explanation aims to shed light on the rationale behind these observations.
The general workflow for IP projects typically involves the following steps:
- Cell lysis to obtain total protein solution and preparation of specific antibodies.
- Mixing specific antibodies with the test sample to form antigen-antibody complexes.
- Enrichment of the mixture using magnetic beads.
- Washing the mixture to remove nonspecifically bound proteins.
- Eluting and separating the protein-antibody complexes from the magnetic beads.
Workflow of the immunoprecipitation (IP) in-solution digestion protocol described (Turriziani et al., 2014)
The experimental steps for all pull-down projects are as follows:
- Preparation of DNA or RNA samples and cell lysates.
- Mixing DNA or RNA samples with cell lysates to form DNA-protein or RNA-protein complexes.
- Enrichment and separation of the complexes using methods such as magnetic beads.
- Washing to remove nonspecifically bound proteins.
- Eluting and separating the protein-antibody complexes from the magnetic beads.
In vitro pulldown assays to study protein interactions with modified histone tails, DNA and assembled nucleosomes (Wierer et al., 2016).
From the outlined processes, it is evident that components such as magnetic bead-protein complexes and elution solutions can be directly submitted for protein identification. If the eluted samples require gel electrophoresis, the resulting gel strips can be submitted for subsequent protein identification.
References
- Turriziani, Benedetta, et al. "On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics." Biology 3.2 (2014): 320-332.
- Wierer, Michael, and Matthias Mann. "Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes." Human molecular genetics 25.R2 (2016): R106-R114.
